The proposed research is planned to provide a comprehensive approach to the identification of cytoplasmic genes, in mammalian cells, involved in cancer as well as in normal cellular processes. Mitochondrial gene mutations will be sought by mutagenic treatments favoring mitochondrial mutations followed by suitable selection. Putative cytoplasmic mutations are being tested by enucleation and cybrid formation or reconstitution, using pairs of cell lines carrying nuclear and cytoplasmic genetic markers. Pairs of related cell lines, one normal and the other transformed or tumorigenic, are being tested by similar methods, enucleation followed by cybrid formation or reconstitution of each nucleus in the cytoplasm of the other to determine whether the tumorigenicity is of nuclear or of cytoplasmic origin. Cell lines being investigated include: Balb/3T3 plus SV40-3T3; cell lines for the Fidler mouse melanoma series selected for increasing metastatic efficiency; virally transformed, chemically transformed, and spontaneously arising tumorigenic cells from a non-transformed diploid laboratory hamster cell line CHEF/18; and mutants induced by treatment with adriamycin and other anti-cancer agents. Cytoplasmic mutations will be studied by genetic recombination, by molecular methods with restriction endonuclease fragments. A product of mitochondrial protein synthesis previously found by us to regulate nuclear DNA synthesis in CHO cells will be further examined with other cell lines in permeabilized cells or with isolated nuclear DNA synthesis.